Peptide bond formation in the presence of triaryl phosphites and heterocyclic disulfides

ABSTRACT

IMPROVED AND COMMERCIALLY ADVANTAGEOUS PROCESS FOR THE PREPARATION OF A PEPTIDE WHICH COMPRISES REACTING A CARBOXYL COMPONENT WHICH IS AN AMINO ACID OR PEPTIDE HAVING FREE CARBOXYL GROUP AND OTHER PROTECTED REACTIVE FUNCTIONAL GROUPS WITH AN AMINO COMPONENT WHICH IS AN AMINO ACID OR PEPTIDE HAVING FREE AMINO GROUP AND OTHER PROTECTED REACTIVE FUNCTIONAL GROUPS IN THE PRESENCE OF A TRIARYL PHOSPHITE AND A DISULFIDE OF A A MERCAPTOHETEROCYCLIC COMPOUND CONTAINING A NITROGEN-CARBON DOUBLE BOND WITH WHICH THE DISULFIDE LINKAGE IS CONJUGATED.

U.S. Cl. 260-1125 Claims ABSTRACT OF THE DISCLOSURE Improved andcommercially advantageous process for the preparation of a peptide whichcomprises reacting a carboxyl component which is an amino acid orpeptide having free carboxyl group and other protected reactivefunctional groups with an amino component which is an amino acid orpeptide having free amino group and other protected reactive functionalgroups in the presence of a triaryl phosphite and a disulfide of amercaptoheterocyclic compound containing a nitrogen-carbon double bondwith which the disulfide linkage is conjugated.

This invention relates to improvements in the preparation of peptides.

More particularly, this invention is concerned with an improved andnovel process for the preparation of a peptide which comprises reactinga carboxyl component with an amino component in the presence of atriaryl phosphite and a disulfide of a mercaptoheterocyclic compoundcontaining a nitrogen-carbon double bond with which the disulfidelinkage is conjugated.

The term carboxyl component as used herein is intended to mean an aminoacid or peptide in which other reactive functional groups capable ofaffecting the present reaction than the carboxyl group required for thepresent reaction are protected with protective groups commonly employedin a peptide synthesis. Also, the term amino componen as used herein isintended to mean an amino acid or peptide in which other reactivefunctional groups capable of attecting the present reaction than theamino group required for the present reaction are protected withprotective groups commonly employed in a peptide synthesis.

Heretofore, we have made numerous attempts to find out new processes forthe preparation of carboxylic acid amides, especially those for thepreparation of carboxylic acid amides, for example, peptides without anyracemization as a side reaction. As a result of our previous studies, ithas been found that a carboxylic acid amide can be prepared by reactinga carboxylic acid with an organic amine or a sulfenic acid amide thereofin the presence of a tertiary phosphine and a disulfide of amercaptoheterocyclic compound containing a nitrogen-carbon double bondwith which the disulfide linkage is conjugated, which has been disclosedand claimed in our copending U.S. patent application No. 90,419, filedNov. 17, 1970.

However, this prior process has a problem to be improved in that thetertiary phosphine, which is considerably expensive, should be employedas a reagent.

Then, as a result of our further studies to develop an inexpensivereagent suitable for the above-depicted carboxylic acid amide, e.g.,peptides preparation, it has now been found that the above problem canbe advantageously settled by employing a triaryl phosphite instead ofthe tertiary phosphine and reacting the carboxyl component with theamino component in the presence of such newly developed reagent, i.e.,the triaryl phosphite, and the disulfide.

United States Patent 0 It is, accordingly, a primary object of thisinvention to provide an improved and commercially advantageous processfor the preparation of a peptide by the use of an inexpensive triarylphosphite reagent.

Other objects and advantages of this invention will be apparent to thoseskilled in the art from the following description.

In carrying out the process of this invention, the reaction may besuitably effected by intimately contacting of the above-mentioned fourreactants, i.e., the amino component, the carboxyl component, thedisulfide and the phosphite in a suitable solvent. The order of additionof four reactants is not critical feature, but it is usual to have fourreactants incorporated into a reaction system all at once or to havedissolved other three reactants than the phosphite and subsequently addthe latter to the resulting solution.

The carboxyl and amino components which may be utilized as startingmaterials in the process of this invention may be any of those aminoacids and peptides commonly employed in a peptide synthesis [Forexample, see Chemistry of Proteins, vol. 1 (1969), ed. by S. Akabori, T.Kaneko & K. Narita, Kyoritsu Shuppan Co. Ltd. Japan]. As explainedhereinabove, the amino and carboxyl components to be employed in thisinvention are those specifically protected amino acids or peptides.

Representative of protectings of other reactive functional groups as setforth above are protecting of amino group with such protective groups:as a substituted or unsubstituted benzoyloxycarbonyl group, a tort.alkoxycarbonyl group, formyl group, trifiuoroacetyl group, anarylsulfonyl group, a triarylmethyl group, a substituted thiccarbonylgroup, o-nitrophenoxyacetyl group, a trialkylsilyl group, an arylidenegroup, acetoacetyl group and the like; protecting of carboxyl group withsuch protective groups as an alkyl ester group, a substituted orunsubstituted benzyl ester group, phthalimidomethyl ester group,benzhydryl ester group, a substituted hydrazino group and the like;protecting of guanidyl group with such protective groups asbenzyloxycarbonyl group, nitro group and the like; and protecting of thenitrogen atom in imidazole moiety and sulfhydryl group with suchprotective groups as benzyloxycarbonyl group, benzyl group and the like,but other protective groups, if utilizable for a peptide synthesis, maybe favorably employed in the process of this invention.

The triaryl phosphites which may be employed in the process of thisinvention are those in which the aryl group is a phenyl or naphthylgroup which may be substituted with a halogen atom, e.g., chlorine,bromine, fluorine and iodine, an alkyl group, advantageously, of 1 to 4carbon atoms, e.g., methyl, ethyl, propyl and butyl, and an alkoxygroup, advantageously, of 1 to 4 carbon atoms, e.g., methoxy, ethoxy,propoxy and butoxy.

In view of commercial availability, preferred examples of the triarylphosphites include symmetric triphenyl phosphite in which the phenylgroup may be substituted with any of the above-depicted substituents,representative of which are triphenyl phosphite, tris(chlorophenyl)-phosphites, tris(methoxyphenyl)phosphites, tritolylphosphites and thelike.

The disulfide which may be employed in the process of this invention isa disulfide of a mercaptoheterocyclic compound containing anitrogen-carbon double bond with which the disulfide linkage isconjugated and it may be symmetric or asymmetric. Representativeexamples of these disulfides include disulfides of a 5- or 6-memberedmercaptoheterocyclic compound having one or more nitrogen atoms as ahetero atom and optionally having as a substituent a halogen atom, e.g.,bromine, chlorine, fluorine and iodine, a lower alkoxy group, e.g.,methoxy,

ethoxy, propoxy and butoxy and a lower alkyl group, e.g., methyl, ethyl,propyl and butyl and condensed ring derivatives thereof: For example,2,2-dipyridyl disulfide, 2,2-dipyrimidinyl disulfide, 4,4-dipyridyldisulfide, 2,2- diquinolyl disulfide, 2,2-dipyrazinyl disulfide,2,2-diquinoxalinyl disulfide, 4,4-diquinazolinyl disulfide, 6,6'diacridinyl disulfide, 2,2-diimidazolyl disulfide, 2,2-dibenzimidazolyldisulfide, 2,2-dibenzothiazolyl disulfide,

2,2 dipurinyl disulfide and 2,2 bis(1-azaazulenyl)disulfide. Substitutedor unsubstituted dipyridyl disulfides are advantageous in view ofcommercial availability.

In the process of this invention may be satisfactorily employed anysolvent that would not adversely affect the present reaction. Examplesof the solvents which may be employed in the process of this inventionare a wide variety of inert organic solvents, for example, ethers, e.g.,dioxane and tetrahydrofuran; halogenated hydrocarbons, e.g., chloroformand methylene chloride; esters of carboxylic acids, e.g., ethyl acetateand butyl acetate; amides, e.g., dimethylformamide anddimethylacetamide; and the like. However, it is desirable in view of thetype of the starting materials employed, solubility of the solvent andother factors to employ any of the above-depicted amides as a solvent inthe present reaction.

The reaction of the process of this invention may be favorably effectedeven in the presence of a minor amount of water.

The reaction temperature in the process of this invention is notcritical feature, but the reaction may be usually and preferablyeffected at room temperature or lower. However, an extremely elevatedtemperature is not advantageous because of possible occurrence of someside reactions.

The reaction period of time i not critical feature, too, and may bevaried mainly depending upon the starting materials and reagentsemployed and the kind of the solvents employed as well as the reactiontemperature applied, but the reaction is usually effected for about 2 toabout 16 hours.

After completion of the reaction, the desired product may be recoveredfrom the reaction mixture by a conventional means. For instance, aftercompletion of the reaction, a suitable solvent in which the desiredproduct is insoluble may be added to either the reaction mixture or theresidue obtained by the removal of the solvent from the reactionmixture, thereby separating out the desired product as crystallinesubstances in situ and then the substances so separated may be recoveredby filtration. The desired product thus recovered may be, if desired,further purified by a conventional means, e.g., recrystallization andcolumn chromatography, but the product thus recovered is frequently of apure form.

The following examples are given for the purpose of illustrating of thisinvention, but they should not be construed to be limiting the scope ofthis invention.

EXAMPLE 1 Benzoyl-L-lucylglycine ethyl ester (1) In 5 ml. ofdimethylformamide were dissolved 467 mg. of benzoyl-L-leucine, 206 mg.of glycine ethyl ester and 485 mg. of 2,2-dipyridyl disulfide and to theresulting solution was added 910 mg. of tris(p-chlorophenyl)phosphite.The resulting mixture was stirred at 30 C. for 15 hours. Thereafter, thesolvent was distilled off from the reaction mixture and the residue wassubjected to a thin layer chromatography utilizing silica gel to give572 mg. of the desired product melting at 154 C. [a] =-32.3 (C.=3.1,ethanol) (2) The same procedure as in the above (1) was repeated exceptthat 683 mg. of triphenylphosphite was employed instead of thetris(p-chlorophenyl)phosphite, thereby yielding the desired product.

(3) The same procedure as in the above 1) was repeated except that 874mg. of tris(p-methoxyphenyl)- phosphite was employed instead of thetris(p-chlorophenyl)phosphite and the reaction was effected at 15 C. for3 hours, thereby yielding the desired product.

(4) The same procedure as in the above (1) Was repeated except that 874mg. of tris(p-methoxyphenyl) phosphite and 683 mg. of2,2-bis(5-nitropyridyl)disulfide were employed instead of thetris(p-chlorophenyl)ph0sphite and the 2,2-dipyridyldisulfide,respectively, and the reaction was effected at 30 C. for 3 hours,thereby yielding the desired product.

(5) The same procedure as in the above (1) was repeated except that 547mg. of 2,2-bis(S-methylpyridyl) disulfide was employed instead of the2,2-dipyridyldisulfide and the reaction was effected at 30 C. for'3hours, thereby yielding the desired product.

EXAMPLE 2 Benzyloxycarbonyl-L-asparaginyl-S-benzyl-L-cysteine methylester In 5 ml. of dimethylformamide were dissolved 533 mg. ofbenzyloxycarbonyl-L-asparagine, 451 mg. of S-benzyl- L-cysteine methylester and 485 mg. of 2,2'-dipyridyldisulfide and to the resultingsolution was added 910 mg. of tris (p-chlorophenyl)phosphite. Themixture was then stirred at 15 C. for 2 hours. Thereafter, 50ml. ofether was added to the reaction mixture to separate out crystallinesubstances, which were then recovered by filtration to give 610 mg. ofthe desired product melting at 196 C. [a] -3l.7 (C. 2.4, pyridine).

EXAMPLE 3 BenZyloxycarbonyl-L-asparaginylglycine ethyl ester In 5 ml. ofdimethylformamide were dissolved 533 mg. ofbenzyloxycarbonyl-L-asparagine, 206 mg. of glycine ethyl ester and 485mg. of 2,2-dipyridyldisulfide and to the resulting solution was added910 mg. of tris(p-chlorophenyl)phosphite. The mixture was stirred at 40C. for 2 hours. Thereafter, 50 ml. of ether was added to the reactionmixture to separate out crystalline substances, which were thenrecovered by filtration to give 680 mg. of the desired product meltingat 184185 C. [041 5.5 (0.: l, dimethylformamide).

EXAMPLE 4 Benzyloxycarbonyl-L-valylglycine ethyl ester In 5 ml. ofdimethylformamide were dissolved 503 mg. of benzyloxycarbonyl-L-valine,206 mg. of glycine ethyl ester and 485 mg. of 2,2-dipyridyldisulfide andto the resulting solution was added 910 mg. oftris(p-chlorophenyl)phosphite. The mixture was then stirred at 40 C. for5 hours. The solvent was distilled off from the reaction mixture underreduced pressure and to the residue was added benzene to separate outcrystalline substances, which were then recovered by filtration to give637 mg. of the desired product melting at 166 C. [a]n= 31.8 (C.=1.0,methanol).

EXAMPLE 5 Benzyloxycarbonyl-L-seryl-L-tyrosine methyl ester In 5 ml. ofdimethylformamide were dissolved 478 mg. of 'benzyloxycarbonyl-L-serine,390 mg. of L-tyrosine methyl ester, 485 mg. of 2,2-dipyridyldisulfideand 681 mg. of triphenylphosphite and the resulting mixture was stirredat 20 C. for 12 hours. The solvent was distilled off from the reactionmixture under reduced pressure and the residue was subjected to a columnchromatography utilizing silica gel to give 5 31 mg. of the desiredproduct melting at 1l5116 C. [a] +13.5 (C. =2, dimethylformamide).

EXAMPLE 6 N benzyloxycarbonyl S-benZyl-L-cysteinyl-L-tyrosyl-L-isoleucyl-L-glutaminyl-L-asparagine tert. butyl ester In 5 ml. ofdimethylformamide were dissolved 501 mg. ofN-benzyloxycarbonyl-S-benzyl-L-cysteinyl-L tyrosine, 429 mg. ofL-isoleucyl-L-glutaminyl-L-asparagine tert. butyl ester, 242 mg. of2,2'-dipyridyldisulfide and 455 mg. of tris(p-chloropheny1)phosphite andthe resulting mixture was then stirred at 15 C. for 12 hours. Thesolvent was distilled ofi from the reaction mixture under reducedpressure and to the residue was added a mixture of methylene chloridewith ether to give crystalline substances. The substances were thenrecovered by filtration and washed successively with the above mixture,0.2 N hydrochloric acid, Water, a 5% aqueous solution of sodiumbicarbonate and then water followed by air-drying. There was obtained732 mg. of the desired product melting at 230 C. M1 20.0 (C.=O.5,dimethylformamide).

What is claimed is:

1. A process for the preparation of a peptide which comprises reactingan amino acid or peptide wherein other reactive functional groups thancarboxyl group are protected with protective groups with an amino acidor peptide wherein other reactive functional groups than amino group areprotected with protective groups in the presence of a triaryl phosphiteand a disulfide of a mercaptoheterocyclic compound containing anitrogen-carbon double bond with which the disulfide linkage isconjugated.

2. The process according to claim 1 wherein said triaryl phosphite isselected from the group consisting of triphenyl phosphite,tris(chlorophenyl)phosphites, tris(methoxyphenyl)phosphites andtritolylpho-sphites.

3. The process according to claim 1 wherein said triaryl phosphite isselected from the group consisting of triphenyl phosphite,tris(pchlorophenyl)phosphite and tris- (p-methoxyphenyl phosphite.

4. The process according to claim 1 wherein said disulfide is selectedfrom the group consisting of substituted or unsubstituted dipyridyldisulfides.

5. The process according to claim 1 wherein said disulfide is selectedfrom the group consisting of 2,2'-dipyridyl disulfide,2,2-bis(5-nitropyridyl)disulfide and 2,2-bis(5- methylpyridyl)disulfide.

References Cited Mukaiyama et al.: Tet. Let., 1970, 1901.

LEWIS GOTTS, Primary Examiner R. J. SUYAT, Assistant Examiner

